Developments in recombinant deoxyribonucleic acid ("DNA") technology have opened up new avenues for medical treatment. The location and sequences of an increasing number of disease-related genes are being identified, and clinical testing of nucleic acid-based therapeutics for a variety of diseases is now underway.
Gene therapy involves the introduction of genetic material into a cell to facilitate expression of a deficient or defective protein. Missing or defective genes (sequences of DNA encoding messenger RNA which are used as templates for protein construction) which are responsible for the production of these proteins result in a class of genetic disease often referred to as `inborn errors of metabolism`. In some cases the disease can be treated by controlling the diet, as in the case of phenylketonuria, in which the liver enzyme responsible for the conversion of phenylalanine to tyrosine is defective. Untreated, this disease can result in mental retardation.
Treatments available for most genetic diseases are not as straightforward as merely altering the diet. For example, adenosine deaminase (ADA) deficiency results from a missing or defective gene that makes the adenosine deaminase enzyme. This enzyme is essential for a healthy immune system. ADA deficiency, however, is the disease successfully treated by the first human "gene transfer" experiment conducted by Kenneth Culver in 1990 (see, Culver, GENE THERAPY: A HANDBOOK FOR PHYSICIANS, MaryAnn Liebert, Inc. publishers, p. 33-40 (1994)).
One method of introducing nucleic acids into a cell is mechanically, using direct microinjection. However this method is only practical for transfecting eukaryotic germline cells for the production of transgenic systems. To be effective in treating a disease, a nucleic acid-based therapy must enter many cells.
Systemic gene transfer entails distributing nucleic acids to target cells and then transferring the nucleic acid across a target cell membrane intact and in a form that can function in a therapeutic manner. In vivo gene transfer is complicated by serum interactions, immune clearance, toxicity and biodistribution.
The in vivo gene transfer methods under study in the clinic consist almost entirely of viral vectors. Although viral vectors have the inherent ability to transport nucleic acids across cell membranes and some can integrate exogenous DNA into the chromosomes, they can carry only limited amounts of DNA and also pose risks. One such risk involves the random integration of viral genetic sequences into patient chromosomes, potentially damaging the genome and possibly inducing a malignant transformation. Another risk is that the viral vector may revert to a pathogenic genotype either through mutation or genetic exchange with a wild type virus.
Lipid-based vectors have also been used in gene transfer and have been formulated in one of two ways. In one method, the nucleic acid is introduced into preformed liposomes made of mixtures of cationic lipids and neutral lipids. The complexes thus formed have undefined and complicated structures and the transfection efficiency is severely reduced by the presence of serum. Preformed liposomes are commercially available as LIPOFECTIN.RTM. and LIPOFECTAMINE.RTM.. The second method involves the formation of DNA complexes with mono- or poly-cationic lipids without the presence of a neutral lipid. These complexes are prepared in the presence of ethanol and are not stable in water. Additionally, these complexes are adversely affected by serum (see, Behr, Acc. Chem. Res. 26:274-78 (1993)). An example of a commercially available poly-cationic lipid is TRANSFECTAM.RTM..
Other efforts to encapsulate DNA in lipid-based formulations have not overcome these problems (see, Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980); and Deamer, U.S. Pat. No. 4,515,736).
Ideally, a delivery vehicle for nucleic acid will be small enough (&lt;300 nm) and stable enough in circulation to distribute from local injection sites or following intravenous injection. The composition will have the maximum amount of nucleic acid per particle and will be homogeneous and reproducible. The composition should also efficiently transfect the target cells.
Surprisingly, the present invention provides such compositions, methods for their preparation, and methods for the introduction of nucleic acids into cells.